The studies proposed will continue our efforts to characterize the actin- based cytoskeleton of the intestinal epithelial cell and its apical brush border (BB). These studies will be pursued with the dual goals of providing general insights into the actin based cytoskeleton of nonmuscle cells, and also to dissect the roles of actin and its associated binding proteins in the physiologic functions of the enterocyte. Our primary focus of study during this grant period will be on the family of unconventional myosins--that is, myosins other than the conventional, two-headed and tailed BB myosin which is expressed in the terminal web domain of the BB. This will include continued biochemical and functional studies on avian BB myosin I. This single-headed myosin, that has as its light chains (lc), multiple calmodulins (CM), forms the bridges which laterally tether the microvillar (MV) actin core to the membrane. Three sets of experiments are proposed. One will investigate the molecular basis for CM interaction with BB myosin heavy chain (hc), as well as the mechanism by which CM regulates the enzymatic, mechanochemical and membrane binding activities of this myosin. A second goal will be to elucidate the regulatory functions of a recently discovered kinase that phosphorylates the tail domain of BB myosin I hc. Finally, we plan to identify and characterize the MV membrane protein that serves as a receptor for BB myosin I. A second major project is based on the discovery that subclones derived from the human intestinal cell line, Caco-2, may express multiple unconventional myosins in addition to BB myosin I. Strategies for the cloning of these myosins are outlined. Once cloned, we plan to probe the function of these myosins in vivo through expression of truncate (headless) myosins in transfected Caco-2 cells. Finally, we outline a pilot study in which we hope to establish the feasibility of applying genetic and molecular genetic approaches to dissecting BB cytoskeletal protein function. To this end we propose the molecular cloning of the proteins which form the cytoskeleton of the Drosophila midgut BB--beginning with the MV core protein villin.
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